Ceramides are dried and dissolved in 20 µl
pyridin then 40 µl benzoyl chloride are added. Heat 1 h at 60°C.
Evaporate the solution under an efficient fume hood and add 4 ml methanol. Heat again 1 h
at 70°C. Evaporate the methanol phase, add 1 ml hexane and 1 ml Na2CO3
saturated methanol solution. Vortex and centrifuge at low speed.
The lower phase is washed first with 1 ml methanol containing 0.6 HCl and then with pure
methanol. The lower phase is evaporated and the residue is dissolved iin a known volume of
hexane before analysis by HPLC.

As a highly specific recombinant ceramide kinase was
cloned, characterized and prepared, a sensitive, rapid and specific enzymatic
method for cellular lipid extracts may be adopted (Bektas
M et al., Anal Biochem 2003, 320, 259). The new ceramide kinase assay is
more specific than the diacylglycerol kinase method and eliminates the need for
analysis of the lipid product by TLC.

Study of the structure of
ceramides

Since ceramides are made of one long-chain base
and one fatty acid, their chemical structure is defined when the pattern of each of these
constituents is determined. A complete knowledge of the ceramide pool is reached only
after identification of all the molecular species forming this pool.

1 ml methanolic HCl is added to dried ceramides
(1-3 mg) in a Teflon-sealed glass tube and the mixture is heated 5 h at 75°C and then
cooled.

A- Extraction and separation of fatty acids

The methanolic solution is washed directly two
times with 2 ml hexane, the washings are collected and evaporated, the lower phase being
kept for long-base extraction. The dry extract contains methylated fatty acids either
normal or hydroxylated.

Separation of the normal and hydroxylated fatty acids:

Fatty acids are separated by TLC on silica gel plates with hexane/diethyl ether (85/15,
v/v) as eluent. Detected after primuline spray under UV light, spots corresponding to the
two fatty acid classes are scraped and fatty acids are eluted with dichloromethane
washings (2 times 2 ml). Standard solutions are prepared by methylation with BF3/methanol
of commercial compounds (one normal and one hydroxylated fatty acid).
Normal fatty acids can be analyzed directly by GLC but hydroxylated fatty acids must be
previously derivatized by reacting with a reagent such as SIL-A from Sigma (100 µl, 15
min at 30°C) or BSTFA-TMCS from Alltech (100 µl, 4-5 h at 20°C).

B- Extraction of long-chain bases

After hexane extraction, the lower methanolic
phase is alkalinized by adding 1 ml 7M NaOH followed by two extractions with 2 ml diethyl
ether.

Long-chain bases are analyzed as previously described
after, if necessary purification by TLC.

Comments :

Another approach to the technical problem of the hydrolysis of
glycosphingolipids has been described using a one-spot heating in a microwave
oven with 0.1 M NaOH in methanol for 2 min followed by 1M HCl in methanol for 45
s (Itonori
S et al., J Lipid Res 2004, 45, 574).

A separation procedure of ceramide molecular species was proposed without the need of
derivatization using the evaporative light-scattering detection (Zhou JY et al, J
chromatogr A 1999, 859, 99).
The effects of the presence of triethylamine and formic acid in the mobile phase
on the evaporative light scattering detection were also reported (Gaudin K et
al. J Liq Chrom Rel Technol 2000, 23, 387).

Procedure:

Column: Kromasil C18 (Eka Nobel) (125x2 mm, 5µm), the column was thermostated at
35°C.
Mobile phase: acetonitrile/propanol (70/30, v/v) containing 10mM triethylamine and 10 mM
formic acid, flow rate: 0.4 ml/min
The response of the detector was not linear but linearization was possible using double
logarithmic coordinates between 10 to 350 ng injected. The lower quantification
limit was about 5 ng of ceramide.
The authors proposed another detection system using a post-column addition of a solution of
1,6-diphenyl-1,3,5-hexatriene enabling a fluorescence detection with a linear response
from 10 to 1000 ng and a similar sensitivity.

SEPARATION OF DERIVATIZED CERAMIDES

Ceramides derivatized with naproxen are separated into molecular species by reverse-phase
HPLC on a C18 stationary phase and with fluorescence detection. The separation is on the
basis of the chain-length and degree of unsaturation of the alkyl moiety considering that
only one type of long-chain base is present. The pattern is more complex if several
long-chain bases are present.

We give below a chromatogram obtained from ceramides prepared from bovine brain
sphingomyelin (Type III from Sigma). Only 100 ng of ceramides were injected.

Peaks are labeled according to the fatty acid moiety using standard commercial species
and a graph relating the logarithm of the corrected retention time and the number of
carbon atoms (linear relationship). Some peaks are unknown.

Gas chromatography procedure

Capillary gas chromatography was used
with success for the quantitative determination of molecular species of
ceramides either free or prepared from sphingomyelins (Tserng
KY et al., Anal Biochem 2003, 323, 84). Not all molecular species were
separated, but all the major molecular species were readily separable and the
nonpolar methylsiloxane column was used for more than 3 years with daily
analyses of biological samples.

Mass spectrometry procedure

A sensitive, selective and rapid method was described for the analysis of ceramides in the
human stratum corneum by direct coupling of HPLC with an electrospray ion-trap mass
spectrometry. This combination enables the differentiation between ceramide species
without special sample preparation.
see: Vietzke J-P et al, Chromatographia 1999, 50, 15.

A direct measurement of ceramide molecular species from lipid extracts was
developed using electrospray ionization tandem mass spectrometry (Han
X, Anal Biochem 2002, 302, 199). A similar technology was used to
appreciate the amount of C18 ceramide in apoptotic mammalian cells (Haynes
TAS et al., Anal Biochem 2008, 378, 80). Individual 2-hydroxy and nonhydroxy
ceramide were readily identified on a 1000-fold linear dynamic range and with a
detection limit at the sufemtomole range. This technique may be directly
applied to chloroform extracts of cellular samples. A similar sensitive
technique coupled with liquid chromatography was described for the analysis of
N-acyl dihydrosphingosines in human hair (Masukawa
Y et al., J Chromatogr A 2006, 1127, 52). A sensitive and accurate
normal-phase liquid chromatography and atmospheric pressure chemical ionization
mass spectrometry method was developed for to profile and quantify all ceramides
(normal and acylated) in mouse skin (Liou
YB et al., Anal Biochem 2010, 401, 107).

OTHER ANALYTICAL METHODS

- High-temperature micro
liquid chromatography for lipid molecular species with evaporative light
scattering detection. Hazotte A et al., J Chromatogr A 2007, 1140, 131-9A quick and simple method is described to analyze the molecular species
of ceramide type III and type IV. Ceramide where analysed using a capillary
column maintained at 150°C with an ethanol–butanol gradient and a flow rate
of 60 ml
min-1. - Quantitation of yeast ceramides using
HPLC and ELSD. Zhou Q et al., J Chromatogr B, 2002, 780, 161-9Phytosphingosine-containing ceramides and ceramide III and IV from Sigma
were separated on a silica column coupled with light-scattering detection. After
a simple saponification step, these compounds were directly and effectively
separated without interference from other molecules (sterols). A 25-min gradient
elution between chloroform and chloroform/ethanol (75/25) was used. Under
optimized HPLC-ELSD conditions, the detection limit was about 0.4 mg,
the optimal amount being about 0.8 mg.

- Analysis of ceramides by
HPLC coupled to tandem mass spectrometry. Fillet M et al., J Chromatogr A 2002,
949, 225-233Ceramides were separated on a
RP C18 column with a mobile phase formed by gradient mixture of water/ACN/2-propanol
and ACN/2-propanol. In less than 12 min, all ceramides were separated, the
linearity confirmed in the range 0-50 ng, le limit of detection being about 0.3
ng.- Structure-retention diagrams of ceramides established for their
identification. Gaudin K et al., J Chromatogr A, 2002, 973, 69-83Molecular species of ceramides analysis was carried out using a porous
graphitic carbon column. The structure-retention diagrams constitute an
identification method using only retention data.